Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14

Abstract

Lipopolysaccharide induces a strong pro-inflammatory reaction of macrophages upon activation of Toll-like 4 receptor (TLR4) with the assistance of CD14 protein. Considering a key role of plasma membrane rafts in CD14 and TLR4 activity and the significant impact exerted on that activity by endocytosis and intracellular trafficking of the both LPS acceptors, it seemed likely that the pro-inflammatory reaction could be modulated by flotillins. Flotillin-1 and -2 are scaffolding proteins associated with plasma membrane rafts and also with endo-membranes, affecting both the plasma membrane dynamics and intracellular protein trafficking. To verify the above hypothesis, a set of shRNA was used to down-regulate flotillin-2 in Raw264 cells, which were found to also become deficient in flotillin-1. The flotillin deficiency inhibited strongly the TRIF-dependent endosomal signaling of LPS-activated TLR4, and to a lower extent also the MyD88-dependent one, without affecting the cellular level of TLR4. In contrast, the depletion of flotillins down-regulated the CD14 mRNA level and the total cellular content of CD14 protein, and decreased the amount of CD14 on the cell surface. The constitutive CD14 endocytosis remained unchanged but CD14 recycling was enhanced via EEA1-positive early endosomes and golgin-97-positive trans-Golgi network, likely to compensate for the depletion of the cell-surface CD14. Notably, a paucity of surface CD14 in resting cells can inhibit TLR4 signaling after the stimulation of cells with LPS. In conclusion, we have shown here that flotillins modulate the pro-inflammatory response of macrophages to LPS by affecting the abundance of CD14.